Oxidative stress affects sperm health and fertility—Time to apply facts learned at the bench to help the patient: Lessons for busy clinicians

Abstract Background Increased oxidative stress (OS), resulting from the delicate balance between reactive oxygen species (ROS) production and antioxidant defense, is closely linked to sperm abnormalities and male subfertility. Elevated ROS levels particularly affect sperm quality. The vulnerability of spermatozoa to ROS is due to the absence of DNA repair mechanisms and the high presence of polyunsaturated fatty acids in their membranes. Methods This article updates and advances our understanding of the molecular damage caused by OS in spermatozoa, including lipid peroxidation, DNA damage, motility, and functionality. Additionally, the review discusses the challenges in diagnosing OS in semen and recommends accurate and sensitive testing methods. Case studies are utilized to demonstrate the effective management of male infertility caused by OS. Main findings Highlighting the need to bridge the gap between research and clinical practice, this review suggests strategies for clinicians, such as lifestyle and dietary changes and antioxidant therapies. The review emphasizes lifestyle modifications and personalized care as effective strategies in managing male infertility caused by OS. Conclusion This review calls for early detection and intervention and interdisciplinary collaboration to improve patient care in male infertility cases related to increased OS.

abnormal sperm morphology, elevated ROS levels are frequently implicated in subfertility or infertility. 2,3 reproductive medicine, there remains a significant disconnect between the detailed laboratory research on OS and its effects on sperm health, and the actual use of this knowledge in clinical practice.Despite significant progress in identifying and treating male infertility, nearly 50% of cases remain unexplained, lacking a clear cause or contributing factor. 4This issue mainly arises from the difficulty in transforming laboratory-based results into practical, patient-focused treatments.The key challenge is to incorporate detailed molecular findings regarding OS and sperm function into concrete clinical procedures.This discrepancy highlights the urgent need for cross-disciplinary teamwork, connecting research with clinical application, to improve fertility results using the deep understanding acquired through laboratory studies.Recent publications by the Global Andrology Forum (GAF) emphasize emerging trends and findings in the field of reproductive health, especially the role of OS in male infertility, 5,6 which strengthens the frontier of knowledge in this field.There is a need to synthesize recent research, highlight the ongoing challenges in translating these findings to therapeutic strategies, and propose actionable steps to integrate these insights into clinical settings.
Thus, this review underscores the importance of understanding the delicate balance between ROS generation and antioxidant defense in male fertility, highlighting the need for translating bench research to clinical practice.

| Basic concept: What is oxidative stress?
OS occurs when there is an imbalance between the production of ROS and the ability of the cellular antioxidant system to neutralize them. 7ROS are highly reactive molecules, typically generated during normal cellular metabolism, and can damage lipids, proteins, and nucleic acids.Excessive ROS production overwhelming cellular antioxidant defenses leads to increased OS, which is linked to various health issues, including male infertility. 8,9S encompasses several forms, including oxygen free radicals like superoxide anions, hydroxyl radicals, and hyperoxyl radicals, along with non-radical forms such as hypochlorous acid and hydrogen peroxide, as well as reactive nitrogen species.These factors play a multifaceted role in male fertility. 4At normal physiological levels, ROS are essential for sperm capacitation, the acrosome reaction, and sperm-egg fusion.Antioxidant defense in seminal fluid is crucial for maintaining sperm health by mitigating the harmful impacts of excessive ROS.This regulation is supported by antioxidants found in the seminal fluid, such as vitamins E and C, taurine, β-mercaptoethanol, cysteine, cysteamine, and hypotaurine. 10However, when ROS levels surpass the scavenging capacity of these antioxidants, OS-induced sperm damage occurs.

| Sources of oxidative stress in the male reproductive system
In human ejaculate, the primary sources of ROS are seminal leukocytes and morphologically abnormal spermatozoa.Residual cytoplasm or cytoplasmic droplets, often containing enzymes like glucose-6-phosphate dehydrogenase (G6PD), significantly contribute to ROS production.
Mitochondrial dysfunction and plasma membrane activity in spermatozoa, along with the enzyme NADPH oxidase 5 (NOX5), exacerbate ROS generation.Seminal fluid leukocytes, such as polymorphonuclear leukocytes and macrophages, are notable ROS producers, particularly when activated by infections or inflammation.Leukocytospermia, identified by the World Health Organization, is a condition marked by an abnormally high concentration of leukocytes in semen.Some of the important endogenous and exogenous sources accounting for OS in the male reproductive system are discussed below.

Radiation
Mobile phone radiation significantly increases ROS in seminal plasma, damaging sperm DNA and affecting sperm motility, count, and vitality. 11,12Both the thermal and non-thermal effects of radiofrequency waves can disrupt spermatogenesis and induce sperm apoptosis (Figure 1). 13,14festyle factors Smoking, a changeable lifestyle choice, substantially disrupts the balance between ROS production and antioxidant defenses, leading to increased seminal leukocyte and ROS levels of 48% and 107%, respectively. 15,16Furthermore, smoking increases the concentrations of toxic elements such as lead and cadmium in semen and blood, enhancing ROS generation and adversely affecting sperm function. 13cohol consumption leads to increased acetaldehyde production, a byproduct of ethanol metabolism, further boosting ROS and reducing the percentage of functional spermatozoa. 13,17xins Increased industrial and domestic pollution introduces more environmental toxins and endocrine disruptors into the immediate surroundings, which can excessively stimulate testicular ROS production and thus OS, adversely affecting sperm morphology and function.[20]

Leukocytes
Peroxidase-positive leukocytes, primarily polymorphonuclear leukocytes (50%-60%), and macrophages (20%-30%) are sourced from the seminal vesicles and prostate gland.During urogenital infections or inflammation, these cells can produce up to 100 times more ROS than usual, enhancing NADPH production through the hexose monophosphate shunt. 21Inflammation also increases proinflammatory mediators concentrations and decreases antioxidant capacity, potentially triggering a respiratory burst leading to OS. 22 Leukocytospermia is defined as the presence of over one million peroxidase-positive leukocytes per milliliter of semen and is linked to significant impairments in sperm function (Figure 1). 23

Immature spermatozoa
During normal sperm maturation of spermatids into mature, motile spermatozoa, excess cytoplasm is extruded by approximately 50%-75% of the total volume of the early spermatids.Disruption in this process results in the retention of excessive cytoplasm around the mid-piece of spermatozoa, impairing their function (excess residual cytoplasm).Immature spermatozoa that retain cytoplasm and exhibit distorted head morphology are thus major contributors to seminal ROS. 24Excess cytoplasm harbors metabolic enzymes like glucose-6-phosphate dehydrogenase (G6PD) and NADPH oxidase, which are crucial for ROS production through NADPH. 25G6PD, in particular, is essential for catalyzing the hexose-monophosphate shunt, facilitating ROS production and OS. 26 Normal spermatozoa also produce ROS through NADPH oxidase (NOX5) in their plasma membrane and NAD(P)H-dependent oxidoreductase (diaphorase) in their mitochondria, which is a key participant in the high-energy Krebs cycle.This cycle primarily facilitates acetate oxidation, generating three NADH molecules from NAD+, which contribute to electron transport in mitochondria, producing a moderate amount of ROS. 27,28

Sertoli cells
0][31] The addition of scavestrogens, synthetic steroidal estrogens with antioxidant properties, can inhibit ROS production in sertoli cells and mitigate ironinduced cell damage. 31,32This finding suggested that under normal conditions, sertoli cells may support spermatogenesis through controlled production of ROS. 31

Varicocele
Varicocele, characterized by venous dilation in the pampiniform plexus with an abnormal blood flow, is a prevalent cause of male subfertility, affecting up to 40% of infertile male partners. 33[35] F I G U R E 1 Impact of oxidative stress on male fertility.(A) Endogenous (varicocele, leukocytes, and immature spermatozoa) and exogenous (lifestyle, radiation, and toxins) sources of ROS lead to oxidative stress which results in (B) lipid peroxidation and DNA damage, resulting in decreased sperm viability and quality.(C) Antioxidants (AntOx) can mitigate these effects, enhancing membrane stability and sperm quality, thus improving fertility.

| OXIDATIVE S TRE SS AND ITS IMPAC T ON S PERM: UPDATED MECHANIS MS
At physiological levels, ROS are necessary for processes such as sperm capacitation, acrosome reaction, and the fusion of spermatozoon and egg.The antioxidant defense in the seminal fluid is vital for preserving sperm health, as it counters the adverse effects of excess ROS.Many molecules and enzymatic systems have important scavenger effects. 10However, when ROS production exceeds the endogenous antioxidant capacity, the increased OS damages spermatozoa (Figure 1).

| Lipid peroxidation and sperm membrane damage
Spermatozoa are uniquely susceptible to ROS-induced damage.7][38][39] Thus, OS leads to lipid peroxidation (LPO), wherein ROS targets the PUFAs in sperm membranes. 40This interaction results in changes in membrane fluidity, a decline in membrane integrity, and ultimately, impaired sperm function.The integrity of the sperm membrane is crucial as it influences sperm motility, an essential factor for successful fertilization. 41Multiple double bonds in PUFAs and a relative deficiency of cytoplasmic antioxidant enzymes in spermatozoa increase the susceptibility to OS. 12 LPO is primarily initiated by the hydroxyl radical (•OH), which targets the vulnerable hydrogen-carbon bonds in the nonconjugated double bonds of sperm membrane lipids, leading to the formation of stabilized free radicals that enhance lipid peroxidation susceptibility. 12pid peroxyl radicals propagate the chain reaction of lipid peroxidation by interacting with conjugated radicals, thus generating lipid hydroperoxides. 8This oxidative process impacts sperm function by oxidizing sulfhydryl groups, decreasing axonal protein phosphorylation, and reducing sperm motility.Furthermore, hydrogen peroxide, another form of ROS, can diffuse into spermatozoa and inhibit crucial metabolic enzymes such as glucose-6-phosphate dehydrogenase (G6PD).Inhibition of G6PD disrupts the pentose phosphate pathway, reducing the production of NADPH necessary for cellular reduction reactions. 12The diminished availability of NADPH impairs the activity of glutathione peroxidase, a critical antioxidant enzyme in spermatozoa that utilizes reduced glutathione to neutralize ROS.Consequently, a reduction in NADPH levels leads to an increase in phospholipid peroxidation, adversely affecting membrane fluidity and further decreasing sperm motility.
Additionally, byproducts of lipid peroxidation, such as malondialdehyde (MDA), serve as biomarkers of oxidative damage in spermatozoa, and are detectable through various biochemical assays. 12The ROS-induced electron loss from sperm membrane lipids further exacerbates LPO, producing mutagenic and genotoxic aldehydes like MDA, 4-hydroxynonenal, and acrolein. 17Elevated ROS levels may also compromise mitochondrial membrane integrity, triggering caspase activation and subsequent apoptosis, thereby perpetuating ROS production, increasing DNA damage, and accelerating apoptotic processes. 42This cascade highlights the critical role of the sperm plasma membrane as a primary target for ROS, underscoring its potential to compromise genetic integrity through cascade signaling mechanisms.

| Oxidative stress on semen parameters
OS plays a significant role in DNA damage within spermatozoa.
ROS are known to cause both single-and double-strand DNA breaks and chromatin crosslinking, leading to genetic anomalies. 43,44Such DNA modifications not only reduce fertilization success rates but also pose risks of transmitting genetic defects to progeny, affecting the health of subsequent generations. 45A significant number of studies have underlined this correlation, providing a novel insight into the etiology of male infertility. 46,47 elevated sperm DNA fragmentation (SDF) rate has been associated with reduced fertilization rates, poor embryo quality, lower pregnancy rates, and a higher risk of early pregnancy loss.An increasing body of evidence points to a robust correlation between seminal OS and SDF. 48One study showed that infertile patients with a high SDF also exhibited increased markers of OS, indicating an underlying link between these two parameters. 46Experimental models have demonstrated that exogenously induced OS leads to an increase in SDF, thereby directly substantiating this association. 49,50Furthermore, interventional studies have shown that the reduction of seminal OS through antioxidant therapy leads to a decrease in the SDF rate, improving overall fertility outcomes.
These studies provide compelling evidence for a positive correlation between seminal OS and SDF.Excessive ROS can inflict base modifications, strand breaks, and chromatin crosslinks, resulting in SDF. 45,48However, the precise molecular pathways underlying this link require further investigation.
In relation to sperm motility and functionality, OS has been observed to adversely impact these critical attributes. 40Specifically, motility is affected by oxidative harm inflicted on the sperm tail and its energy source, thereby hindering its capability to progress toward and penetrate the oocyte. 51The spermatozoon, specifically its structural components such as the axoneme and the acrosome, exhibits a high susceptibility to oxidative damage instigated by ROS. 40The axoneme, which is essential for sperm motility and primarily composed of microtubules, is particularly vulnerable to OS. Elevated ROS levels can lead to lipid peroxidation of the membrane surrounding the axoneme, compromising its structural integrity and potentially altering its functionality.Such oxidative impairment can reduce sperm motility, thereby impeding the ability of the sperm to navigate the female reproductive tract to reach and fertilize the oocyte. 52reover, the acrosome reaction, which occurs in the acrosome located at the anterior region of the spermatozoon, is critical for penetration through the protective barriers of the oocyte.This reaction involves the release of enzymes essential for fertilization. 53wever, OS can disrupt this finely tuned process by either prematurely triggering or completely inhibiting the acrosome reaction, consequently hindering effective adhesion and penetration of the oocyte. 54The dual role of ROS, as both essential signaling molecules and damaging agents, underscores the importance of maintaining a balanced oxidative state to preserve sperm functionality and optimize male reproductive potential. 14mparative studies have consistently revealed that men with elevated OS levels exhibit markedly poorer sperm health than men with lower OS levels.This manifests as a decrease in sperm count, motility, and viability, and an increase in the SDF rate. 43,51,52,55,56The association between heightened OS and diminished sperm quality highlights the significance of maintaining an oxidative balance for optimal male reproductive health.

| Genetic and epigenetic modifications
OS significantly affects both the genetic and epigenetic integrity of spermatozoa, which in turn influences early embryo development. 57is results in SDF, chromatin structural abnormalities, and a decline in overall sperm quality, including motility and fertilization potential. 48The epigenetic modifications induced by OS in spermatozoa are also crucial for understanding the developmental outcomes of the embryo. 58,59Research has shown that spermatozoa exposed to oxidative conditions can lead to a significant developmental arrest at the stage of embryonic genome activation. 51,60This process has been observed through various experimental studies, including those using animal models like cattle, where it was noted that embryos fertilized with spermatozoa exposed to OS displayed major developmental delays. 61,62These changes in the sperm epigenetic landscape, such as modifications in DNA methylation patterns and histone configurations, do not necessarily correlate directly with the levels of DNA damage, indicating that the epigenetic reprogramming mechanisms might be independently sensitive to oxidative conditions. 57,59rthermore, the introduction of antioxidants has been suggested as a potential therapeutic approach to mitigate this oxidative damage, thus preserving both the genetic and epigenetic integrity necessary for successful fertilization and early embryo development. 63This finding suggests a pivotal role for targeted antioxidant therapies in improving reproductive outcomes in patients with OSinduced infertility.

| CLINI C AL S TUD IE S AND RE S E ARCH DATA 4.1 | Research studies
The OS plays a critical role in determining sperm quality and is intimately connected to the reproductive potential across various animal species.8][69] This inverse relationship is often attributed to lipid peroxidation of sperm membranes.Oxidative damage reduces membrane fluidity, impacting sperm motility and ultimately its fertilizing capacity. 66,70Moreover, LPO has been identified as a detrimental factor that decreases sperm quality by impairing its motility and vitality. 71,72Mitochondrial functionality, which is essential for providing the energy necessary for sperm motility, is also affected by OS.Studies indicate that ROS can cause mitochondrial dysfunction by damaging the mitochondrial DNA, leading to decreased mitochondrial membrane potential (MMP) and is lower than the normal MMP by −80 to −120 mV and by altered electrochemical gradient to reduced ATP production, essential for all the energy-dependent processes for sperm motility. 64In B. taurus, for instance, there is a positive correlation between MMP and sperm motility, suggesting that mitochondrial health is a critical determinant of motility and, by extension, fertilization capability. 73Interestingly, the response to OS and the resultant impact on sperm function appear to be species-specific.While some species exhibit a direct negative impact of increased ROS on sperm quality, others show varying degrees of resilience or adaptation to oxidative conditions, which might reflect evolutionary adaptations to environmental OS. 64,69For example, in B. taurus, the relationship between MMP and motility underscores the species-specific energy metabolism strategies that spermatozoa employ to maintain functionality despite oxidative challenges. 69,735][76] It has been reported how in vitro handling and manipulation of spermatozoa during assisted reproduction technology (ART) procedures can generate OS, which adversely affects sperm function.The research has indicated that spermatozoa experience increased ROS production during various in vitro procedures, such as washing, centrifugation, and cryopreservation.This OS is linked to sperm DNA damage, which can lead to reduced fertilization rates and compromised embryo development. 51,76Furthermore, specific impacts observed in in vitro settings, such as decreased sperm motility and vitality due to oxidative modifications induced by handling and environmental stressors, have also been reported.For instance, the centrifugation process used in sperm preparation for intracytoplasmic sperm injection (ICSI) and other ART techniques is particularly highlighted for exacerbating oxidative conditions, thereby increasing the likelihood of sperm DNA fragmentation.The resulting oxidative DNA damage in spermatozoa is critical because it holds potential implications for the success rates of ART outcomes, including lower pregnancy rates and increased risks of miscarriage. 51,77These findings underscore the delicate balance required in in vitro environments to manage OS, highlighting the need for optimized protocols that minimize oxidative damage to spermatozoa, thereby preserving their functional integrity and enhancing the chances of successful fertilization and healthy embryo development.The ongoing challenge in ART-related procedures is to refine and apply methods that reduce OS, such as antioxidant supplementation or gentler handling techniques, to improve overall reproductive outcomes.
Furthermore, omics studies in the realm of male fertility have illuminated the profound impacts of molecular mechanisms on sperm quality, offering a multidimensional understanding that extends beyond traditional assays.Through the comprehensive integration of omics datasets, Park et al. 78 identified distinct molecular pathways governing male fertility in boars and bulls, highlighting speciesspecific responses to fertility challenges.This study identified key differences in gamete production and protein biogenesis-associated pathways in bulls with below-normal fertility, suggesting a linkage between impaired protein synthesis during spermatogenesis and fertility outcomes.Conversely, boar spermatozoa with normal fertility exhibited enriched mitochondrial-associated metabolic pathways, indicative of optimized energy metabolism contributing to better reproductive outcomes. 79Furthering the discourse, recent omics approaches have enabled the profiling of spermatozoa at an unprecedented scale, with studies identifying fertility-related molecular markers that differentiate between varying fertility levels.
These investigations not only enhance the understanding of sperm biology but also pave the way for novel diagnostic tools and therapeutic strategies aimed at improving male reproductive health.For instance, comparative omics analyses have highlighted the crucial role of mitochondrial functionality in sperm motility and overall fertility, demonstrating that the integrity of mitochondrial processes is critical for maintaining the energy supply required for effective sperm function and fertilization. 78,80These insights from omics studies are reshaping the understanding of sperm quality and fertility, emphasizing the importance of molecular mechanisms in determining reproductive success.The integration of transcriptomic, proteomic, and metabolomic data offers a holistic view of the biological functions influencing sperm quality, which is vital for developing targeted interventions aimed at enhancing male fertility across different species.

| Clinical studies
Excessive production of ROS occurs physiologically in several circumstances, including lifestyle factors-such as alcohol consumption, cigarette smoking, and obesity-or the presence of varicocele, exposure to radiation, taking medications, and so on. 81Various clinical studies have documented the negative impact of seminal OS on sperm quality and male fertility since Aitken and colleagues first reported ROS in washed human semen using a chemiluminescence assay. 82S can damage sperm ultrastructure leading to peroxidation of membrane lipids, proteins, and DNA, consequent to cellular apoptosis when its levels exceed the cellular scavenger capacity which is greatly reduced in spermatozoa. 21[85] This evidence has led to the coining of the acronym "MOSI" (male oxidative stress infertility), recently proposed to indicate those patients whose infertility is attributable to high levels of seminal OS. 86 More specifically, a negative correlation between seminal OS levels and the percentage of spermatozoa with normal motility was outlined in a prospective clinical study on 39 infertile patients and 13 fertile controls.The high seminal OS has also emerged in patients with teratozoospermia with a higher percentage of spermatozoa with amorphous heads, damaged acrosomes, midsection defects, cytoplasmic remnants, and tail defects, suggesting that sperm morphology is a good indirect index of seminal OS. 83 More recently, seminal OS was negatively correlated with sperm concentration and motility in a study of 847 patients. 87e high seminal OS has also been associated with poor ART outcomes and failure of embryo development in clinical settings. 88SDF, an indirect measure of the effects of OS on spermatozoa, has been associated with pregnancy outcomes.In particular, a meta-analysis of 56 studies reported the negative impact of this parameter on the outcomes of ART (both IVF and ICSI). 89SDF has also been found to be associated with unexplained recurrent miscarriages (RPL) 90 and the latest guidelines from the European Society for Human Reproduction and Embryology (ESHRE) on the management of RPL mention that SDF assessment can be considered for diagnostic purposes in couples with RPL. 91spite sporadic attempts to find seminal ROS cut-off values predictive of ART outcome, 92 to date no threshold has been introduced into clinical practice, mainly due to measurement limitations (see Section 5.2 for details).

| Semen analysis
The conventional analysis of sperm parameters, such as sperm count, morphology, and motility, offers clinicians a surrogate metric for evaluating seminal OS, with asthenozoospermia posited as a particularly reliable indicator of OS.An increase in seminal plasma viscosity is associated with elevated levels of MDA, a marker of lipid peroxidation, and a concomitant decrease in antioxidant capacity within the seminal plasma. 93Moreover, infections with Ureaplasma urealyticum in semen are linked to increased seminal plasma viscosity and enhanced generation of ROS. 93The presence of an excessive number of round cells in semen may suggest leukocytospermia, a known contributor to elevated ROS production.To differentiate these cells from immature spermatozoa, additional diagnostic assessments, such as peroxidase tests, seminal elastase measurement, or immunostaining for the cluster of differentiation 45 (CD45), a leukocyte-specific transmembrane glycoprotein, are recommended.
Notably, abnormal sperm morphology and the presence of cytoplasmic droplets are indicative of dysfunctional spermatozoa prone to unregulated ROS production.Furthermore, compromised integrity of the sperm membrane, assessable through the hypo-osmotic swelling test, is associated with the presence of OS. 13

| Total antioxidant capacity
To evaluate the total antioxidant capacity (TAC) within seminal plasma, luminol is utilized as a chemiluminescent probe.This assay is calibrated against Trolox, a water-soluble analog of vitamin E, ensuring the standardization of measurements.The results are expressed in terms of a ROS-TAC score, which quantifies the cumulative antioxidant activities contributed by all constituents, including vitamins, lipids, and proteins. 94

| Evaluation of ROS via chemiluminescence
The quantification of ROS in seminal fluid is typically conducted using a chemiluminescence assay (Figure 2).This technique involves the utilization of a luminometer coupled with a chemiluminescent substrate, specifically luminal (5-amino-2,3-dihydro-1,4-phthalazine dione; Sigma-Aldrich, St. Louis, MO, USA).To prepare the samples, semen is initially liquefied and then centrifuged at 300 g for 7 min.
The resultant seminal plasma is aliquoted and stored at −20°C for later TAC measurement.The remaining sperm pellet is washed with phosphate-buffered saline (PBS, pH 7.4), and re-suspended in PBS to a concentration of 2 × 10 6 sperm/mL for the measurement of basal ROS levels.For the assay, a control reaction is set up using 10 mL of a 5 mM solution of luminol in 400 mL of PBS.Luminol, prepared as a 5 mM stock solution in dimethyl sulfoxide, is added to the sperm suspension to serve as the chemiluminescent probe.The reaction mixtures are then incubated within the luminometer for 15 min to facilitate the quantification of ROS levels.Luminol is sensitive to both extracellular and intracellular ROS, detecting these species through the emission of light upon reaction with the radicals.This emitted light is converted into an electrical (photon) signal by the luminometer, and the resultant data is expressed in relative light units per second per 10 6 sperm.In assays involving washed sperm suspensions, normal ROS concentrations typically range from 0.10 to 1.03 × 10 6 counted photons per minute per 20 × 10 6 sperm. 94

| Lipid peroxidation markers
In spermatozoa, the accumulation of lipid peroxides leads to the formation of various degradation products, notably MDA, acrolein, hydroxynonenal, and isoprostanes.These compounds serve as biomarkers of OS and can be quantitatively assessed. 95Among these biomarkers, MDA is most commonly measured using the thiobarbituric acid (TBA) assay.This assay exploits the interaction between MDA and TBA to form a 1:2 adduct, which is a colored complex.The concentration of this complex can be determined using fluorometric or spectrophotometric techniques. 13,95

| Oxidation-reduction potential in seminal fluid
The oxidation-reduction potential (ORP), also referred to as redox potential, quantifies the electron transfer capacity between chemical F I G U R E 2 Different methods of measurement of seminal oxidative stress.entities, encapsulating the dynamic balance between oxidants and reductants (Figure 2).This parameter is instrumental in assessing the OS within biological systems. 96Technological advancements have enabled the use of a galvanostatic method to monitor electron flux, which proves useful in gauging OS changes post-trauma or during intense physical activity. 97Integrating ORP evaluation with conventional semen analysis aids in identifying the origins of poor semen quality and male infertility.The MiOXSYS System (Male Infertility Oxidative System; https:// mioxs ys.com/ mioxs ys-system/ ), employing ultra-high impedance electrometry, measures semen ORP by assessing electron exchange between antioxidants and oxidants present. 98Unlike alternative methodologies, the MiOXSYS technique requires neither specialized training for operation nor specific sample preparation protocols.It allows for the ORP determination from a minimal volume (30 μL) of fresh or thawed samples within approximately 4 min, maintaining result stability for up to 120 min post-collection. 98If analysis post this timeframe is impractical, sample cryopreservation is recommended.

| Challenges and limitations in the diagnosis
The introduction of the measurement of seminal ROS levels into clinical practice has been severely slowed down by the limitations of the tests currently in use.The sixth edition of the World Health Organization (WHO) semen analysis manual introduced ROS evaluation tests in the "advanced examination" section, which collects tests (e.g., luminol, ORP, and TAC) that do not have sufficient validation evidence and therefore are recommended only in a research context.Thus, their interpretation in the clinical setting requires a certain caution degree. 99spite being the first method introduced for measuring ROS, chemiluminescence requires a lot of time and expensive equipment, and, above all, the results are highly variable.Sperm age, volume, centrifugation, temperature control, and background luminescence can interfere with the measurement, thus explaining the high intraindividual variability in the test. 100,101TAC has long been used to estimate the total antioxidant capacity, but is limited by the expensive equipment required, the time of inhibitory activity, and does not provide information on the levels of antioxidant enzymes that play an important scavenger role. 101,102Tests for measuring MDA, an indirect indicator of high levels of OS at the seminal level, require rigorous controls, are not specific, and only provide post-hoc measures. 101P has recently proven to be an attractive option for ROS assessment, amenable to standardization in the future.Some evidence suggests the reproducibility and reliability of the MiOXSYS in measuring ORP.ORP levels have been negatively correlated with sperm concentration, sperm motility, normal morphology, and total motile sperm count 103 and positively with SDF rate. 46,103,104A cutoff value of 1.34 (mV/10 6 sperm/mL) was recently proposed to discriminate between good and poor-quality sperm, with a positive predictive value of 94.7%. 98,105However, sample viscosity can still pose a challenge, being a source of intra-individual variability in ORP assessment. 101

| Recommendations for best practices in clinical settings
As indicated in the WHO manual for semen analysis, 99  Accordingly, the SDF has been included in the latest edition of the WHO manual for semen analysis, in the "Extended examination" section, in which all tests that should not be performed routinely in clinical practice, but which can be required for diagnostic purposes in specific situations. 99Among all currently available tests for measuring SDF, the TUNEL test, the sperm chromatin dispersion test, the Comet test, and the acridine orange (AO) test are described in the manual. 99erall, the guidelines of the main scientific societies such as the American Society for Reproductive Medicine (ASRM), the American Urological Association (AUA), the ESHRE, and the Italian Society of Andrology and Sexual Medicine (SIAMS), agree in not requiring the SDF test as a first-level examination during the management of infertile patients. 91,106,107An orderly and sequential diagnostic process (of course starting from a detailed medical history) is essential for trying to understand the causes of infertility.The SDF test should be requested only in cases where conventional work-up shows negative results and a clear etiology cannot be diagnosed.
The ASRM/AUA and ESHRE suggest (while the European Urological Association [AUA] recommends) SDF testing in couples with RPL from natural conception or ART, as well as in men with unexplained infertility. 75,91,106

| Antioxidant therapy: Types, effectiveness, and potential risks
Antioxidants such as vitamins C and E have been shown to improve sperm quality by protecting spermatozoa from oxidative damage, thereby enhancing their motility. 108,1091][112][113] Moreover, antioxidants help maintain the integrity of sperm DNA, reducing the risk of compromised embryo development and miscarriages. 114However, antioxidant supplementation is not without risks.Excessive intake can lead to pro-oxidative effects, which increase OS and can harm sperm health. 115Some antioxidants may also interact with medications, potentially reducing their effectiveness or leading to adverse reactions. 116The optimal dosages of antioxidants for improving male reproductive health remain unclear, with both insufficient and excessive intakes posing potential risks. 116,117The consumption of certain micronutrients and compounds has been thoroughly examined for their possible benefits in reducing OS in the male reproductive system. 1181.1 | l-carnitine and acetyll-carnitine l-carnitine and acetyll-carnitine, both derivatives of the amino acid lysine, play crucial roles in the oxidation of mitochondrial fatty acids.In relation to male fertility, these compounds are vital for maintaining proper sperm morphology and motility.119,120 High levels of ROS can impair sperm functionality.The administration of lcarnitine and acetyll-carnitine has been shown to enhance sperm motility by reducing oxidative damage and improving mitochondrial function.121,122

| Zinc and folic acid
Zinc, a vital trace mineral, is essential for a variety of physiological functions including DNA synthesis, RNA transcription, and cellular metabolism.Folic acid is key for DNA synthesis and repair.In male reproductive health, deficiencies in either nutrient can diminish sperm quality. 123The combined supplementation of zinc and folic acid has been shown to increase sperm count in men with reduced fertility, suggesting a synergistic effect that may protect against oxidative damage to sperm DNA. 124

| Vitamin E and selenium
Vitamin E, a lipid-soluble antioxidant, and selenium, a trace element, are both powerful antioxidants essential for preventing oxidative damage in sperm, thereby enhancing motility and overall sperm health.Their combined supplementation has shown greater efficacy in improving male reproductive health than taking either nutrient alone. 125,126

| Coenzyme Q10
CoQ10 is a critical component of the mitochondrial electron transport chain, essential for energy production, and also acts as an antioxidant. 112,127Deficiency in CoQ10 can impair sperm motility due to reduced energy production and increased OS.Supplementation has been shown to improve sperm parameters by mitigating oxidative damage and enhancing energy production in spermatozoa. 111,127spite the favorable outcomes reported in numerous studies, some findings indicate minimal or no improvement in sperm parameters following antioxidant supplementation.These discrepancies may be due to differences in study designs, sample populations, types and dosages of antioxidants, and duration of supplementation. 116As researchers continue to explore potential treatments for male infertility, they find increasing evidence that antioxidants and dietary modifications can play significant role in improving reproductive health and fertility outcomes.They offer a practical approach to combating OS, a significant detriment to male reproductive health.However, careful application is essential.Extensive, ongoing research is needed to determine optimal antioxidant amounts and combinations, and understanding individual responses to these treatments will be crucial for tailoring personalized therapies.While the associated risks are generally low compared to the potential benefits, medical approaches should be based on thorough diagnostic evaluations and scientifically sound guidelines. 128

| Lifestyle and dietary modifications
Research has demonstrated that improvements in semen quality can be achieved through targeted dietary changes and regular exercise, independent of body mass index changes.0][131] Animal studies further suggest that these lifestyle changes can positively affect embryo development and offspring metabolic health. 132Adhering to Mediterranean-style diets-rich in fruits, vegetables, seafood, and antioxidant-laden plant foods-also correlates with superior semen quality. 133,134 terms of micronutrients, carotenes, ascorbic acid, tocopherols, selenium, zinc, l-arginine, and CoQ10 are particularly beneficial for male fertility. 135Moderate exercise is advisable for improving fertility and mitigating OS, though intense exercise may be detrimental. 1361][142] Caffeine should be limited to the equivalent of 3 cups of coffee daily, 143 and cannabis use is discouraged for managing male infertility. 144The potentially harmful effects of anabolic steroids on the male hypothalamic-pituitary-testicular axis could involve the use of gonadotropins, selective estrogen receptor modulators, and aromatase inhibitors.However, the use of these substances in an off-label manner is not well-researched. 145fective management of psychological stress through meditation, yoga, and similar practices can improve male fertility. 146,147Further investigation is necessary to define the precise benefits of stress reduction techniques and therapeutic approaches like cognitive behavioral therapy.It is also vital to manage stress related to sexual performance to enhance fertility outcomes.Sufficient sleep appears to play a crucial role in enhancing semen quality, as suggested by research. 148,149onetheless, the specific lifestyle parameters and their optimal levels remain undefined and warrant additional research.

| Emerging therapies and future directions in treatment
Emerging therapies and future directions in the treatment of OS in male reproductive health focus on advancing current methodologies and exploring innovative approaches. 150The continuous development of more targeted antioxidant therapies is a prime area of interest.Novel antioxidants and compounds that specifically target mitochondrial function and reduce ROS production are under investigation. 151,152ese could offer more precise mechanisms for protecting spermatozoa against oxidative damage.Additionally, gene therapy presents a promising frontier.Research is aiming to correct genetic defects that contribute to increased OS or compromised antioxidant defenses in spermatozoa. 150,153Techniques such as CRISPR/Cas9 offer the potential for directly repairing these genetic anomalies, thereby enhancing sperm quality and overall reproductive health. 153Nanotechnology is another emerging field that could play a significant role in treating male infertility related to OS. Nanoparticles can be engineered to deliver antioxidants directly to specific cells or tissues, potentially increasing the efficacy and reducing the side effects associated with the systemic administration of antioxidants. 154,155Furthermore, the role of the microbiome in male reproductive health is gaining attention.Studies suggest that modulating the gut microbiome could influence systemic antioxidant levels and immune responses, indirectly impacting OS levels and fertility. 156As the understanding of the biochemical pathways involved in male fertility deepens, personalized medicine approaches are becoming more feasible.These would involve comprehensive genomic, proteomic, and metabolomic profiling to tailor specific antioxidant therapies to individual needs, enhancing both effectiveness and safety. 157Thus, while antioxidants and lifestyle modifications currently offer significant benefits in managing OS in male reproductive health, the field is evolving.Future therapies are likely to be more precise and personalized, addressing the underlying causes of OS with greater accuracy and fewer side effects.Extensive research and clinical trials will be essential to validate these innovative approaches and integrate them into standard practice.However, ethical considerations of genetic diagnostics and emerging technologies, like CRISPR, include potential long-term effects, unintended consequences, and psychological impacts.Responsible integration requires preventing misuse, ensuring informed consent, equitable access, and psychological support.

| C A S E S TUD IE S S HOWC A S ING SUCCE SS IN IMPROVING S PERM HE ALTH AF TER REDUCING OXIDATIVE S TRE SS
The patient, a 32-year-old male, presented with his 28-year-old partner to the fertility clinic with concerns about difficulty conceiving.

| Highlighting key takeaways that can be applied in clinical settings
9][160][161][162] According to Mayorga-Torres, increased intracellular ROS production and DNA fragmentation have been observed in infertile patients compared to fertile men, whereas no significant differences were observed in conventional sperm parameters between fertile men and infertile patients.Furthermore, OS-induced DNA damage in spermatozoa of male infertile patients may have implications for the health of children conceived in vitro. 163

| Interventions and solutions for busy clinicians
Addressing male infertility related to high levels of ROS and therefore OS is a nuanced area in which andrologists can play a pivotal role.The intervention strategies include two fundamental aspects, eliminating or reducing as much as possible all the causes of increased OS and increasing the levels of substances with antioxidant activity.
As regards the first aspect, interventions must be aimed at eliminating, where possible, all diseases that cause an increase in OS (e.g., urogenital infections, obesity, varicocele, etc.) and lifestyle changes.The latter can be recommended to the patient at the time of the first visit and includes stopping cigarette smoking, drinking alcohol, using narcotics, and so on.The patient should also be advised to avoid, if possible, occupational exposure to toxins that can increase ROS levels (e.g., industrial chemicals, pesticides, etc.).
As regards the second aspect, the patient can be advised, after careful evaluation of his diet, to increase the intake of foods richer in antioxidants.Along this same line, the opportunity for treatment with antioxidants should also be discussed with the patient.It has indeed been shown that the prescription of these supplements improves sperm quality and patient fertility by reducing OS, although with a low level of evidence.
It is essential to choose the molecule/s and the dosage of antioxidants based on the results of the laboratory tests performed on the patient so that the prescription is as compliant as possible with the pathophysiological aspects present in the patient.Indeed, it is useful to remember that although antioxidants are effective if appropriately prescribed, individual responses can vary significantly.
Therefore, the treatment strategy should be personalized based on a thorough assessment of the patient's general health, lifestyle, and specific fertility concerns.Doctors should also stay up to date on the latest research and clinical guidelines in this evolving field to provide the best possible care for their patients.

| Diagnostic tests available for measuring oxidative stress in semen samples
The assessment of OS in semen samples is a critical aspect of male infertility diagnosis, hence for a successful treatment.However, the current diagnostic tests have limitations (see Section 5.2).
Castleton 164 reported that the MiOXSYS® and OxiSperm® II assays, while included in the WHO manual, did not provide additional clinical utility beyond standard semen analysis.Overall, the absence of significant associations between nitroblue tetrazolium (NBT)-reactivity and measurements of sperm function or OS suggests the limited diagnostic potential of the MiOXSYS and OxiSperm II assays.Agarwal 165 suggested that the ORP test could be a cost-efficient and sensitive option for measuring OS in semen.
Tunc 166 developed a standardized protocol for the NBT assay, which is effective in identifying sperm OS.Gosalvez 158 highlighted the need for an inexpensive and easy-to-perform assay to detect OS in semen.
Overall, the measurement of OS in the semen fluid is of great relevance for a proper diagnosis.However, further research is of pivotal importance in this area.

| Recommendations for reducing oxidative stress
In the realm of reproductive health and male infertility, the use of antioxidant supplements is a topic of significant interest, especially in cases where high ROS production and hence increased OS have been diagnosed.Excessive weight has been linked to reduced sperm production but also to higher OS.Therefore, diet and daily exercise need to be planned appropriately.A deficiency of nutrients, particularly zinc, selenium, and vitamin C, may disturb sperm production.Therefore, it is important to have a healthy and balanced diet.Proper treatment following the doctor's instructions and daily exercise boosts the immune system and normalizes the situation. 167Furthermore, infection, inflammation, and other diseases eventually present and capable of increasing the levels of OS must be treated with their specific therapeutic approaches.Supplementation can be used if the diet lacks the required amounts of nutrients with antioxidant properties.Currently, despite the effectiveness of antioxidant administration in improving conventional sperm parameters and pregnancy rate, 6 there is no generally accepted agreement on the best supplementation therapy, either as a single compound or as a mixture of them.Furthermore, the level of evidence of the various studies published in the literature is classified as low or moderate quality, due to the lack of standardized therapeutic regimens widely used in these studies and the lack of common inclusion criteria for the male population undergoing treatment. 6The acceptance of antioxidant supplements for treating male infertility varies globally.In some countries, these supplements are widely used and recommended, while in others, they are prescribed with more caution due to a lack of compre- However, it is crucial to note that the efficacy and safety of these supplements can vary and over-supplementation can sometimes have adverse effects.

| Importance of early detection and intervention
Understanding the role of ROS in male infertility and recognizing specific OS markers will enable clinicians to tailor treatments that target the underlying oxidative damage.This potentially results in reversing sperm abnormalities and increasing the chances of successful conception.Early intervention in patients with high ROS levels addresses immediate fertility issues, as it enables targeted interventions such as lifestyle modifications and antioxidant therapy, to overcome OS.However, accurate diagnostic methods also help prevent long-term reproductive health complications, emphasizing the importance of routine screening for OS markers in male fertility assessments.This is particularly important in cases of male subfertility or idiopathic infertility, and probably even more given a history of RPL.

| Encouraging interdisciplinary collaboration between research scientists and clinicians for optimal patient care
Encouraging interdisciplinary collaboration between researchers and clinicians is vital to optimize patient care in cases of male infertility attributed to high OS, as this collaboration fosters the integration of cutting-edge scientific insights with clinical expertise, leading to better outcomes and personalized treatment strategies.In particular, the standardization of reliable and reproducible tests to measure OS is urgently needed.This problem can be solved and rapidly introduced into clinical practice with continued strong collaboration between basic scientists and clinicians.

| FUTURE D IREC TI ON S IN RE S E ARCH AND CLINI C AL PR AC TICE
The future directions in OS research and clinical practice involve several critical advancements and shifts in focus to enhance male reproductive health.A significant area of future research will likely be the development and integration of advanced diagnostic tools that can accurately and non-invasively assess OS levels in semen.Such tools will be crucial for the early detection of oxidative damage, allowing for timely interventions that could significantly improve male fertility outcomes.Additionally, studies using omics technologies to uncover new biomarkers and therapeutic targets are expected to further elucidate the molecular pathways influenced by OS.
On the clinical front, personalized medicine will become increasingly important.Treatments tailored to individual OS profiles and genetic backgrounds are expected to significantly improve patient outcomes.This approach will leverage insights gained from advanced genomics and proteomics studies, enabling clinicians to design antioxidant therapies that are more effective and have fewer side effects than current options.Furthermore, interdisciplinary collaboration between researchers, clinicians, and technologists will be essential to translate these findings from bench to bedside rapidly and efficiently.The integration of artificial intelligence and machine learning in diagnostic and treatment processes could also play a transformative role, offering new ways to manage and treat OS-related male infertility.

| CON CLUS ION
This review has underscored the pivotal role that OS plays in male infertility, providing clinicians with a deeper understanding of how bench research translates into clinical practice.Key takeaways for clinicians include the importance of early detection and management of OS, as highlighted by the molecular intricacies and pathological consequences discussed.Clinicians are encouraged to adopt advanced diagnostic tools and consider antioxidant therapies alongside lifestyle and dietary modifications to improve patient outcomes.The integration of bench research into clinical settings, particularly in the field of male fertility, has the potential to significantly enhance patient care.This review not only bridges the gap between theoretical research and practical application but also emphasizes the necessity for ongoing interdisciplinary collaborations.Such endeavors will enable the development of targeted therapies that mitigate oxidative stress and improve sperm quality, thus addressing the underlying causes of male infertility and enhancing reproductive outcomes.

ACK N OWLED G M ENTS
The authors would like to thank Dr. Rosella Cannarella for her help with some sections of this article.
current tests for direct measurement of ROS levels should be avoided in clinical practice, until further validated in large multicenter cohort doubleblind studies.To date, the only test that has demonstrated sufficient reliability and reproducibility and has obtained the consensus of several companies are the tests that measure SDF, which represents an indirect measurement of seminal OS.
The couple had been trying to conceive for the past 18 months without success.The male partner reported a generally healthy lifestyle (Mediterranean diet, regular physical activity, and no alcohol or drug use) but admitted to experiencing high levels of stress at work as a software engineer in a high-stress environment.He referred to smoking 10 cigarettes a day for 10 years.He denied any history of significant medical conditions, including diabetes mellitus, and had not undergone any surgeries.Uneventful physical and genital examination revealed normal-sized and firm testes (right testicular volume: 20 mL and left testicular volume: 18 mL), and no varicocele, hydrocele, or other abnormalities were confirmed via high-resolution ultrasound.The laboratory test results included a normal complete blood count, liver and kidney function test results, and normal endocrinologic assessments, including insulin resistance.In two repeated semen analyses, there was a mild oligoasthenoteratozoospermia without leukocytospermia but elevated levels of SDF rate at the TUNEL test (10%), with local laboratory cut-off normal values <4% and elevated ROS in semen according to the MiOXSYS test, with a value of −5.7 mV/10 6 spermatozoa/mL.The patient was therefore diagnosed with MOSI in the absence of other identifiable causes.His management plan consisted of lifestyle modification with counseling on stress management techniques and advised to quit smoking.The prescribed medical treatment consisted of daily antioxidants (including vitamin C, vitamin E, carnitine, Zn and Se, and CoQ10) together with dietary modifications to include antioxidant-rich foods.In the follow-up semen analysis after 3 and 6 months, there was an improvement in conventional semen parameters, SDF rate, and normalization of the MiOXSYS value to 1.2 mV/10 6 spermatozoa/mL.This case highlights the impact of OS on male fertility, particularly in the context of a modern lifestyle characterized by high stress and suboptimal habits.OS can lead to sperm dysfunction by damaging the sperm membrane and DNA, resulting in decreased fertility.Management focuses on identifying and mitigating contributing factors, antioxidant therapy, and supportive care.
hensive and well-designed clinical trials.Regulatory agencies like the FDA in the United States or the EMA in Europe have different standards and guidelines for supplement use, which impacts global acceptance.Insert here the results and ref of the GAF Survey.